Two of the most important measures of the hematologic status of an individual are the whole blood count and leukocyte differential. Whole blood counts and leukocyte differentials involve the discrimination and then counting of the various cellular components of blood. The various components of blood that may appear in any one sample include red blood cells (RBC), platelets and nucleated cells. In the latter category, a number of different types of leukocytes exist which include lymphocytes (both B and T cells, Natural Killer (or "NK") cells and various subsets thereof), monocytes and granulocytes (including neutrophils, eosinophils and basophils in all maturational stages). Because of the wide possible range of cell types and the range of maturational stages of any one cell type, obtaining whole blood counts and leukocyte differentials often is a difficult and complex procedure in the normal individual but it is more difficult and complex in an abnormal individual. This complexity is emphasized even more when the analysis of bone marrow samples is attempted.
Traditionally, whole blood counts (i.e., number of cells per standard unit of volume) and leukocyte differentials (i.e., number of cells of a given type per standard unit of volume) have been performed manually by counting a small volume of cells using a light microscope and then multiplying the number counted by a factor to account for the volume. Because of both the sample size measured and the level of skill required to distinguish among cell types, a high and often unacceptable degree of variability may be introduced by this method.
Recently, instrumentation has been devised to obtain whole blood counts or leukocyte differentials without having to resort to manual microscopic examination. Currently available instrumentation detect cells either electronically by aperture impedance (e.g., Coulter Counter.TM., described in Coulter, Proc. Nat. Electronics Conf., 12:1034 (1956)) or optically by light scattering and absorption (e.g., Technicon H-6000.TM., described in Breakell et al., Blood Cells, 11:257 1985)). In these instruments, the red blood cell fraction must be separated from the leukocytes and each measurement is taken independently of the other. This must be done because RBC generally outnumber leukocytes by at least 1000:1 in the normal patient. In the abnormal patient (e.g., leukopenic patent), this ratio may be substantially higher.
Other means to obtain leukocyte differentials also exist. Flow cytometers, which are generally described in U.S. Pat. Nos. 4,661,913, 4,284,412 and 3,826,364, and in an article by Herzenberg et al., Sci. Am., 234:108 (1976), have been used to identify different populations of leukocytes in a heterogeneous sample by detecting multiple independent parameters on individual cells that pass through the sensing region. Typically, these parameters include forward light scatter (FLS which is a measure of relative particle size), orthogonal light scatter (OLS which is a measure of relative granularity) and fluorescence. Fluorescence may be measured from cells that incorporate a nucleic acid stain or may be measured from cells bearing surface markers which are labelled with monoclonal antibodies (MAbs) conjugated directly or indirectly to fluorochromes, as described, for example, in U.S. Pat. No. 4,520,110. Separate channels within the flow cytometer sense and record each of the various cell measurements. By combining and comparing these parameters, the various leukocyte components may be distinguished. U.S. Pat. No. 4,727,020 provides one example of how a flow cytometer may be used in this method to obtain leukocyte differentials from blood. This approach, however, is limited to leukocyte differentials and only those obtained from blood.
Taken together, there is no single, standard, accurate instrumentation or methodology currently available that allows a single sample of blood or bone marrow to be analyzed such that all the various cell types present in blood and bone marrow may be discriminated and then counted.